Journal of Reproductive Immunology, 17 (1990) 155-165 Elsevier Scientific Publishers Ireland Ltd.

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JR1 00648

Interleukin- l/3, but not interleukin- lcu, induces acute inflammation-like changes in the testicular microcirculation of adult rats Anders

Bergha and Olof S6derb

“Department of Pathology, University of Umea and bPediatric Endocrinology Unit, Karolinska Hospital, Stockholm (Sweden) (Accepted for publication 10 November 1989)

Summary

A number of inflammatory mediators and cytokines were injected locally into the testis of adult rats in order to test their ability to induce leukocyte accumulation and increased vascular permeability (as studied by a carbon labelling technique). Human recombinant interleukin-l/3 (IL-@) caused increased vascular permeability and leukocyte migration. All the other factors studied - histamine, serotonin, bradykinin, interleukin-la (IL-la), and a partially purified interleukin-la-like factor (tIL-1) from rat testis - did not induce any acute signs of increased vascular permeability or inflammatory response after local injection. It is suggested that local production of IL-l/3 from testicular macrophages could be responsible for the inflammation-like changes that are seen in rat testes after treatment with human chorionic gonadotrophin. Key words: testis; interleukin la; testicular interleukin-l-like factor; interleukin-l/3; vascularpermeability; rat.

Introduction

Treatment of adult rats with luteinizing hormone (LH) or human chorionic gonadotrophin (hCG) induces inflammation-like changes in the testicular microcirculation (for review see Bergh et al., 1988). Four hour8 after treatment, polymorphonuclear leukocytes accumulate in testicular venules, 0165.0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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inter-endothelial cell junctions are opened and by this route macromolecules leak and leukocytes migrate into the interstitial space. The volume of testicular interstitial fluid and the rate of lymph flow are also increased after hCG treatment (Setchell and Sharpe, 1981). The accumulating leukocytes apparently play a role in mediating these microvascular changes as the hCGinduced increase in venular permeability is inhibited in leukocyte-depleted rats (Widmark et al., 1987). The mechanism by which LH/hCG treatment induces inflammation-like changes is, however, unknown but activation of Leydig cells is necessary as an inflammation-like response is not noted after hCG treatment in rats lacking Leydig cells as a result of ethylene dimethane sulphonate (EDS)-treatment (Damber et al., 1987). Interestingly, recent studies have shown that the testis produces large amounts of an interleukin-1 (IL-I)-like factor (tIL-1) (Kahn et al., 1987). Local injection of IL-l into the skin results in an acute inflammatory response, an effect partly mediated by leukocyte binding to endothelium (Dinarello, 1984, 1988; Cotran, 1987; Mantovani and Dejana, 1989). Against this background it is of interest to study the effects of IL-1 on the testicular vasculature. Materials and Methods Animals, carbon-/abelling, tissuefixation and microscopy Adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital(40 mg/kg). The scrotum was opened bilaterally and one testis was injected with the drug (see below) dissolved in 50 ~1 and the contralateral testis was injected with 50 ~1 of the solvent (0.9% saline), using a 27G injection needle. In some of the experiments the tested drug and the solvent was also injected into epididymal fat or into the dorsum of the foot (drug on one side and solvent on the contralateral) in order to verify that the drug was bioactive. At various times after treatment the animals were injected intravenously with a solution of colloidal carbon (Pelikan Drawing Ink, 1 ml/kg) as described previously (Bergh et al., 1987). One hour after carbon injection the rats were killed, the testes were removed and fixed by immersion in a fixative containing 4% formaldehyde, 3% glutaraldehyde and 0.05% picric acid in 0.05 M cacodylate buffer (for details see Bergh et al., 1987). The testes were sliced using a razor blade and the sections were examined for the presence of carbon leakage using a dissection microscope as described previously (Bergh et al., 1987). Parts of the testes were cut into small pieces, postfixed in 1% 0~0, and embedded in epoxy resin (Polybed 812). Testicular morphology was studied in 1 pm thick sections stained with Toluidine Blue.

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Drug treatments The following substances were injected into one testis (and epididymal fat or skin) and vehicle in the contralateral organ. All experiments were performed in triplicate (three animals for each drug and each time-point studied). (a) Human recombinant interleukin-Ifi (hrIL-la, 1000 units/ml, Genzyme, MA, U.S.A.) in a dose of 5 or 50 units. One unit (U) of IL-l is defined by the manufacturer as the amount needed to double the incorporation of tritiated thymidine into cultured murine thymocytes employed as an IL-l bioassay (Oppenheim et al., 1976). The rats were then injected with carbon at 1.5 h or 7 h after treatment and studied as described above. When IL-l is injected locally in the skin the peak response was noted somewhere between 1 and 6 h and 50 U caused a maximal or near maximal effect (Wankowicz et al., 1988; Mason and Van Epps, 1989). In a separate experiment some adult male rats were injected i.p. with ethylene dimethane sulphonate (EDS, 75 mg/kg dissolved in dimethyl sulphoxide : water 1 : 3 v/v). EDS is a Leydig cell toxin that within 3 days results in the total depletion of Leydig cells from the rat testis (Kerr et al., 1985). Three days after EDS treatment one testis was injected with 5 U IL-la and the effect was studied after 1.5 h by carbonlabelling as described above. (b) Human recombinant interleukin-la (hrIL-la, 1000 U/ml, Genzyme, MA, U.S.A.) in a dose of 5 or 50 U. One unit (U) is defined as described for IL-la. The rats were injected with carbon at 1.5 or 7 h after treatment and studied as described above. (cl Testicular interleukin-Z-like factor (tIL-1) purified from rat testis. An aqueous extract was prepared from pooled rat testes as described previously (Kahn et al., 1987). The material was further purified by chromatography on a Polybuffer Exchanger 94 column (Pharmacia, Uppsala, Sweden) and by HPLC gel permeation as described by Gustafsson et al. (1988). The bioactivity of all IL-1 preparations was verified by analysis in murine thymocyte cultures essentially as described previously (Seder and Madsen, 1988; Gustafsson et al., 1988). The amount of tIL-1 injected corresponds approximately to 5 IL-l/3 units (see above). The rats were injected with carbon 1.5,4 or 7 h after drug treatment and studied as described above. (d) Histamine (Histamine HCL, Sigma) in a dose of 50 pg. Serotonin (SHT-creatinine sulphate, Sigma) in a dose of 5 pg. Bradykinin (Sigma) in a dose of 5 pg. The animals were injected with carbon immediately after drug treatment. When these substances are injected into other tissues they cause an acute (within 1 h) increase in vascular permeability (opening of interendothelial cell gaps in post-capillary venules) and doses approximately lo50 times lower than used in the present study result in a maximal response (Movat, 1985).

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Results

Effect of saline In the dissection microscope it was observed that a small expansion of the interstitium (never larger than 1 x 2 mm in size) was created by the injected saline and carbon-leaking vessels were observed in the wall of this cavity (Fig. la). This was found principally in the testes examined within 2.5 h after

Fig. 1. (A) Stereomicroscopic view (12 x magnification) of testicular tissue 1 h after intra-testicular injection with 504 saline. Colloidal carbon was injected iv. immediately after the testis injection. No carbonleakage is observed in the testis tissue, except in close relation (small arrows) to the cavity formed by the injection (large arrows). A 270 needle was inserted secondarily into the tissue section in order to demonstrate its size (diameter 410 pm). A similar picture with occasional carbon leakage only around the injection site was seen after local injection of histamine, serotonin, bradykinin, IL-la, or tIL-1. (B) Stereomicroscopic view (12 x ) of the testicular tissue (far away from the injection site) in a rat given an intra-testicular injection with 50 U IL-l/I. Colloidal carbon was injected 1.5 h after IL-l treatment and the animal was killed after an additional hour. Some of the numerous black stained carbon-leakage sites are indicated with small arrows.

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treatment, suggesting that the injected fluid is fully resorbed after that time. Light microscopy of tissue from the injection site revealed occasional tubule damage and numerous carbon-labelled blood vessels. However, in all other parts of the testis no carbon leaking blood vessels could be observed. A similar response to saline injection with carbon-leakage close to the injection site but not anywhere else in the testis was observed also in the EDS-treated animals . Effect of IL-@ In the testes injected with 5 or 50 U hrIL-la, numerous carbon-labelled blood vessels were detected already in the dissection microscope in all parts of the testes (Fig. lb). The response was of similar magnitude 1.5 and 7 h after treatment. The testicular blood vessels contained numerous polymorphonuclear leukocytes and some had migrated into the interstitial space and large sub-endothelial deposits of carbon particles were observed in post-capillary venules (Fig. 2a). HrIL-10 induced a similar response in EDStreated Leydig cell depleted testes (Fig. 2b). Effect of IL-la In the testes injected with hrIL-la it was possible to find some leaking blood vessels in relation to the injection channel as in the saline injected testes, but in other parts of the organ leukocyte accumulation or leaking blood vessels were not observed (Fig. 2~). There was no difference between the testes examined at 1.5 or 7 h after treatment and both doses were ineffective. Carbon-leakage was, however, observed in the epididymal fat pad and in subcutaneous tissue 1.5 h after injection of 50 U IL-la. Effect of tIL-I In the testes injected with 5 U tIL-1 no leukocyte accumulation or vascular leakage was observed at 1.5, 4 or 7 h after treatment, except for the slight leakage induced close to the injection channel. Injection of 50 ~1 of the tIL-1 preparation did, however, induce some carbon leakage in subcutaneous tissue (Fig. 3) but the response was weaker than after injection with pure hrILla. Effect of histamine, serotonin and bradykinin In the testes injected with histamine, serotonin or bradykinin no vascular leakage could be observed but carbon leakage was sometimes noted in the fat between the testis and epididymis (data not shown). When injected locally in the epididymal fat or subcutaneous tissue, these substances induced a clear cut inflammatory response (carbon leakage but no leukocyte accumulation) as previously shown in several other tissues (Movat, 1985).

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Fig. 2. Light micrographs from rats where the testis was injected locally with 50 U hrlL-l/3 (A), 5 U hrILIfi (B), and 50 U hrIL-la (C) (1000 x magnification). Colloidal carbon was given 1.5 h after the injection and the animals were killed after one additional hour. The rat shown in Fig. B was given EDS 3 days prior to the experiment to remove Leydig cells. No carbon leakage or PMN-leukocyte accumulation was induced by IL-la treatment (C) but IL-l/3 induced leukocyte accumulation and migration and carbon leakage (small arrows) both in testes with (A) and without (B) Leydig cells.

(large arrows)

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Fig. 3. Stereomicroscope views (20 x magnification) of the subcutaneous tissue of the dorsum of the foot in a rat injected with 50 ~1saline on one side (A) and 50 ~1of a solution containing partially purified tIL-1 (B). Colloidal carbon was injected 1.5 h after this treatment and the animal killed after an additional 1 h. Local injection of tIL-1 induced carbon leakage (arrows) in small blood vessels but this was not observed after saline injection.

Discussion We have recently observed that stimulation of the rat testis with hCG/LH ‘results inflammation-like changes in the testicular microcirculation (Bergh et al., 1988). As it is unlikely that hCG or androgens have direct inflammationlike effects on testicular blood vessels (see discussion in Bergh et al., 1988; Sowerbutts et al., 1986), we have suggested that activation of Leydig cells

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results in the local secretion of an inflammation mediator(s) in the testis. The nature of this factor(s) is unknown but it must be leukotactic, be able to open inter-endothelial cell gaps in post-capillary venules (that can be labelled with carbon), and to be effective within 4 h after treatment. The testis is apparently less responsive than other organs to the common acute effects of inflammatory mediators such as IL-la (present study), leukotriene B4 (Stenson et al., 1986), bradykinin (Sowerbutts et al., 1986; present study), histamine (Gabbiani et al., 1970; Kilzer et al., 1985; Sowerbutts et al., 1986; present study) and prostaglandins (Sowerbutts et al., 1986), but the reason for this remains unknown. Sowerbutts et al. (1986) suggested that serotonin (5hydroxytryptamine) could be involved in the hCG-induced increase in vascular permeability, but in this study we did not observe any acute effect of locally injected serotonin and seretonin is not leukotactic (Movat, 1985). It can, of course, not be excluded that these mediators may have other effects on the testicular vasculature, or at other time points, than those yet studied. Some vascular leakage can, however, be induced in the testis by the trauma caused by local injection, indicating that the testis, like other organs, acutely releases mediators causing increased vasclar permeability in response to tissue damage, but the nature of this factor(s) is unknown. It is obviously not of Leydig cell origin as a reaction was observed by saline also in EDS-treated animals. The observation that local dilatation of the interstitial space and vascular leakage can be induced by saline injection indicates that local injection of test substances into the testis may not be a valid method when the aim is to detect moderate effects on testicular vascular permeability. It has recently been claimed that the normal composition of testicular interstitial fluid can be monitored using a push-pull cannula inserted into the testis (Maddocks and Setchell, 1988) but whether this procedure results in increased vascular permeability close to the cannula is not known. An IL-l-like factor is produced locally in the testis, presumably in the seminiferous tubules (Kahn et al., 1987; Syed et al., 1988; Seder et al., 1988, 1989). Its functional role is unknown but it has been suggested that it could serve as a mitogen for germ cells (Seder et al., 1988, 1989, Pollanen et al., 1989). One of the biological roles of IL-l in general is to promote leukocyte binding to the endothelium and to facilitate leukocyte migration (Cotran, 1987; Dinarello, 1988). TIL-1 is similar in structure to IL-la (Kahn et al., 1987; Gustafsson et al., 1988). TIL-1 or IL-la, despite the fact that they were effective in other organs, did not promote any acute inflammatory effects in the testis in the present study. Further, in a recent study by Pollanen et al. (1989), no testicular inflammation was noted after three daily intra-testicular injections of 30 U hrIL-la (unpublished observations). The reason why testicular blood vessels are less sensitive that other tissues to the

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common pro-inflammatory effect of IL-la (Movat and Cybulsky, 1987; Wankowicz et al., 1988) is unknown. It can, however, be argued that as an IL-la-like molecule is produced constitutively in large amounts it is perhaps necessary to have local inhibitors present to avoid continuous inflammation. IL-l inhibitors have indeed been described in other tissues (Larrick, 1989) and there are observations suggesting that such inhibitors, for example amelanocyte stimulating hormone, are also produced in the testis (Boitani et al., 1986; Pollanen et al., 1988). In contrast to IL-la, IL-l/3 induced leukocyte accumulation and increased vascular permeability in the rat testis and doses as low as 5 U were effective. The response was similar to that seen in other tissues after local injection of IL-l/3 (Wankowicz et al., 1988; Movat and Cybulsky, 1987). IL-l/3 has recently been found to stimulate (Verhoeven et al., 1988) or to inhibit (Calkins et al., 1988a; Fauser et al., 1989) Leydig cell steroidogenesis, but, interestingly, IL-la had little if any effect (Calkins et al., 1988b; Fauser et al., 1989). In the present study a vascular response to IL-la was observed also in testes lacking Leydig cells as a result of EDS treatment, indicating that the effect was not mediated via Leydig cells. The observations that IL-la and /I may have different effects in the testis is somewhat surprising since they are generally assumed to act via the same receptor and to share effects in various test systems (Dinarello, 1988), but there are other tissues in which IL-la and fi have different biological effects (Bendtzen et al., 1986; Uehara et al., 1987). The testicular interstitium contains numerous resident macrophages and their cell size is changed after hCG treatment (Bergh, 1987). HCG stimulation causes an increase in IL-l activity in the testis but whether this is related to increased tIL-1 activity or induction of IL-l/I-like material which is not constitutively produced in the testis is unknown (Seder et al., 1989). IL-l/3 is the major (> 9OYo)IL-l protein produced by activated macrophages. As ILl/I is (at present) the only pro-inflammatory substance with demonstrably clear cut acute effects on testicular blood vessels and as this cytokine is indeed able to mimic the inflammatory effects induced by hCG/LH treatment (Bergh et al., 1988), it is not unlikely that IL-l/I, or closely related factor(s), could be involved in the physiological/pathophysiological control of testicular blood vessels. Acknowledgements This study was supported by grants from the Swedish Medical Research Council (projects 5935, 8282), The Maud and Birger Gustavsson, Magnus Bergwall and King Gustav V Jubilee Foundations. Skilful technical assistance was given by Mrs. Sigrid Kilter.

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Interleukin-1 beta but not interleukin-1 alpha, induces acute inflammation-like changes in the testicular microcirculation of adult rats.

A number of inflammatory mediators and cytokines were injected locally into the testis of adult rats in order to test their ability to induce leukocyt...
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